Microsomal cytochrome P-450 has been purified from neonatal pig testis. The enzyme is homogeneous according to electrophoresis on polyacrylamide gels with SDS and to double diffusion and immunoelectrophresis against an IgG fraction from rabbits immunized against the pure enzyme. The enzyme is prepared and stored in the detergent Emulgen (0.02%) and shows a molecular weight of 59,000 with a single NH2 terminal amino acid (methionine) and a heme content of 10 nmoles/mg protein. The pure enzyme shows two activities: 17alpha-hydroxylation and C17,20 lyase activity with both delta 4 (progesterone) and delta 5 (pregnenolone) substrates. The former activity is competitively inhibited by the corresponding 17-alpha-hydroxysteroid. Photochemical action spectra reveal that the heme moriety is involved in both 17alpha-hydroxylation and lyase activity. The 17alpha hydroxy steroids were identified as obligatory intermediates in the side-chain cleavage of C21 steroids. The lyase activity was found to be competitively inhibited by estrogens.